Sensitive and Label-Free Fluorescent Detection of Transcription

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Sensitive and Label-Free Fluorescent Detection of Transcription Factors Based on DNA-Ag Nanoclusters Molecular Beacons and Exonuclease III-Assisted Signal Amplification Bingzhi Li,† Lei Xu,† Yue Chen,‡ Wanying Zhu,† Xin Shen,† Chunhong Zhu,† Jieping Luo,† Xiaoxu Li,† Junli Hong,† and Xuemin Zhou*,† †

School of Pharmacy, Nanjing Medical University, Nanjing 211166, People’s Republic of China Department of Nutrition and Food Safety, School of Public Health, Nanjing Medical University, Nanjing 211166, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: Transcription factors (TFs) regulate gene expression by binding to regulatory regions, and their dysregulation is involved in numerous diseases. Thus, they are therapeutic targets and potential diagnostic markers. However, widely used methods for TFs detection are either cumbersome or costly. Herein, we first applied DNA-Ag nanoclusters molecular beacons (AgMBs) in TFs analysis and designed an assay based on the switchable fluorescence of AgMBs. In the absence of TFs, a single-stranded DNA functioned as a reporter is released from a double-stranded DNA probe (referred as dsTFs probe) under exonuclease III (Exo III) digestion. Then, the reporter triggers downstream Exo IIIassisted signal amplification by continuously consuming the guanine-rich enhancer sequences in AgMBs, resulting in significant fluorescent decrease eventually. Conversely, the presence of TFs protects the dsTFs probe from digestion and blocks the downstream reaction to keep a highly fluorescent state. To testify this rationale, we utilized nuclear factor-kappa B p50 (NF-κB p50) as a model TFs. Owing to the amplification strategy, this method exhibited high sensitivity toward NF-κB p50 with a limit of detection of 10 pM, and a broad linear range from 30 pM to 1.5 nM. Furthermore, this method could detect multiple TFs in human colon cancer DLD-1 cells and reflect the variation in their cellular levels after stimulation. Finally, by conducting an inhibition assay we revealed the potential of this method for screening TFs-targeted drugs and calculating the IC50 of corresponding inhibitors.

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radioisotopes. Western blot and ELISA are popular methods for analyzing proteins. However, specially manufactured chromatin immunoprecipitation (ChIP)-grade antibodies with the ability to recognize TFs/DNA complex are needed when they are adopted in TFs analyses, and these antibodies are the most expensive materials in such detection.11,12 In recent years, emerging assays for TFs detection include fluorescent,13−15 colorimetric,16−18 and electrochemical methods.19−21 Among all these methods, fluorescent assays are attracting accumulating attention due to high sensitivity. Heyduk’s group pioneered fluorescent detection of TFs using two DNA duplexes each containing half of a TFs binding sequence.1 These two duplexes were labeled with fluorescent donor and acceptor, respectively, and the presence of TFs caused the association of two duplexes to generate a fluorescence resonance energy transfer (FRET) signal change.

ranscription factors (TFs) are DNA-binding proteins that play pivotal roles in cell development, differentiation, and growth by regulating gene expression.1,2 Mounting evidence indicates that the dysregulation of TFs is involved in numerous pathological processes. For example, the excessive and persistent activation of nuclear factor kappa B (NF-κB) is implicated in autoimmune diseases, cancers, and viral infections.3−5 Another example is p53, whose abnormal expression is found in more than 50% of cancer cases, and the inactivation of it causes rapid growth of tumor.6,7 Therefore, TFs are not only important indicators in basic medical researches, but also potential diagnostic markers in clinical medicine.8−10 Classical methodologies for TFs analyses include electrophoretic mobility shift assay (EMSA), DNA footprinting, Western blot, and enzyme-linked immunosorbent assay (ELISA). EMSA and DNA footprinting are gel-based methods based on the affinity between TFs and double-stranded DNA (dsDNA) binding sequence. Their drawbacks include the need for well-trained operators, cumbersome work, and the use of © XXXX American Chemical Society

Received: January 6, 2017 Accepted: June 16, 2017

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DOI: 10.1021/acs.analchem.7b00055 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

and elucidating its feasibility, this work not only filled in the blank of applying DNA-AgNCs in TFs quantification, but also proposed a promising detection platform for resolving TFsrelated biomedical issues.

However, the steric hindrance caused by TFs-DNA binding might affect the proximity between donor and acceptor and lead to low FRET signal.22 To increase FRET signal, a variety of fluorescent probes were developed, including conformational change-based TFs beacons,23 Holliday junction structure-based DNA strand exchange probe,24 and signal translation-based molecular beacon probe.25 Nonetheless, the use of costly fluorescent labels make them unsuitable for large-scale detection. Since graphene oxide (GO) can absorb singlestranded DNA (ssDNA) and quench most of fluorophores, it was fused with molecular beacons to realize half-labeled26 or label-free27 detection of TFs, but the unspecific absorption between GO and biomolecules in biological media challenged the sensitivity of such nanosensors.28,29 Some dsDNA specific dyes were integrated with rationally designed DNA probes to implement label-free detection of TFs, but they suffered from either limited sensibility caused by the nature of dyes30,31 or the need for complicated annealing and deactivation processes.32 Hence, there are still great needs for exploring new TFs detection methods with high sensitivity, low cost, and high specificity.33 DNA-Ag nanoclusters (DNA-AgNCs) are a class of nanomaterials with small size (diameter of ca. 2 nm) and bright fluorescence.34,35 By mixing DNA template with silver salts and reducing agent in proper buffer, a nucleation reaction causes the formation of AgNCs at cytosine-rich nucleation sequence.36 DNA-AgNCs are not only brighter and more photostable when compared with organic dyes, but also smaller, less toxic, and less prone to blinking problems when compared with quantum dots.37−39 While one of the most intriguing properties of this material is the switchable fluorescence. Yeh et al. found that the fluorescence of DNA-AgNCs had a 500-fold of enhancement when placed in proximity to guanine-rich enhancer sequences (GRS),40 which provided a powerful biosensing approach by manipulating the proximity-separation state of DNA-AgNCs and GRS.41−45 Inspired by this discovery, Wang’s group proposed a delicately designed DNA-AgNCs molecular beacon (AgMBs) as a versatile biosensor, which placed the DNAAgNCs and GRS at different ends of a hairpin-shaped DNA probe.46 This design endowed the probe with the similar feature of molecular beacons (MBs), which produced distinct signal change upon hybridization with target DNA. Despite that great development has been hitherto made, the bioanalytical application of DNA-AgNCs is mainly focused on detecting nucleic acids and developing aptamer-based sensors,37,41 where we believe that the application scope of DNA-AgNCs could be further explored. In this contribution, we used NF-κB p50 as a model TF and proposed a label-free fluorescent method for sensitive detection of NF-κB p50 based on AgMBs and exonuclease III (Exo III). According to the best of our knowledge, it is the first study that applies nanocluster-based materials in TFs analyses. By utilizing Exo III digestion, we translated the binding between NF-κB p50 and dsNF-κB p50 probe into ssDNA (named as reporter) releasing. Then, AgMBs was manipulated to detect the reporter with the assistance of Exo III recycling signal amplification. Notably, this signal amplification strategy is deeply based on the features that the changeable fluorescence of AgMBs is regulated by an unmodified and digestible DNA. Besides, this homogeneous method simply needs only two steps of sample addition and no complicated temperature regulation, thus, has the potential for being employed in high-throughput detection and drug screening. Overall, by designing an analytical method

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EXPERIMENTAL SECTION 2.1. Materials. HPLC-purified oligonucleotides used in this research were purchased from Sangon Biotech (Shanghai, China). DNA sequences are listed in Table S1 in Supporting Information. AgNO3 (metal basis, 99.999%) and NaBH4 (analytical grade) were obtained from Aladdin Reagent (Shanghai, China). The molecular biology grade salts used for preparation of buffers were purchased from Basic Bioscience Inc. (Shanghai, China). Electrophoresis grade reagent used in native polyacrylamide (native-PAGE) gel are obtained from Macklin Chemical (Shanghai, China). Purified human recombinant NF-κB p50 (untagged) and NF-κB p65 (untagged) were purchased from Enzo Life Sciences (NY, U.S.A.). Exo III and 10× Exo III reaction buffer were provided by Thermo Fermentas (Lithuania). (−)-Dehydroxymethylepoxyquinomicin ((−)-DHMEQ) was purchased from MedChem Express (NJ, U.S.A.). Ultrapure water were produced by a Millipore’s Synergy system equipped with a Biopack filter (MA, U.S.A.) and treated with DEPC before use. 2.2. Synthesis of AgMBs. AgMBs was synthesized according to former literature.40,46 In a typical procedure for synthesizing 2 mM AgMBs, 1 OD of template DNA lyophilized powder was dissolved by 976 μL of sodium phosphate buffer (10 mM Na2HPO4/NaH2PO4, 100 mM CH3COONa, 10 mM Mg(CH3COOH)2, pH 7.4). This solution was heated to 90 °C for 10 min and then slowly annealed to room temperature for at least 1 h. Then, 10 mM of fresh-prepared AgNO3 solution was added to hairpin and aged in a dark place. NaBH4 solution was added to the reaction system, followed by vigorous shaking for 30 s. The final concentration ratio between hairpin, AgNO3 and NaBH4 was 1:6:6. The reaction was kept in dark place at 4 °C overnight before use. 2.3. Quantification Assay and Fluorescence Measurement. The 0.5 mg/mL (10 μM) stock solution of NF-κB p50 (MW: ∼50 kDa11,47) provided by manufacturer was diluted using buffer (10 mM Na2HPO4/NaH2PO4, 10% glycerol, pH 7.4). For NF-κB p50 detection, 100 nM of dsNF-κB probe was mixed with various concentrations of NF-κB p50 diluted solution and 10 μL of protein binding buffer (10 mM Na2HPO4/NaH2PO4, 100 mM CH3COONa, 10 mM Mg(CH3COOH)2, 10% glycerol, 0.05 mg/mL poly(dI-dC) (Thermo scientific, U.S.A.), pH 7.4). Then, 2 μL (10 U/μL) of Exo III and 3 μL of 10× Exo III reaction buffer were added into the solution to digest dsDNA and release reporter. It should be noticed that ultrafiltration centrifugation was conducted using Amicon Ultra 10K device (Millipore, Ireland) to remove the DTT in Exo III before use. Afterward, AgMBs (10 μL, 1 μM), DEPC-treated water (8 μL), and 10× Exo III reaction buffer (2 μL) was added to the reaction and incubated in a thermocycler (Applied Biosystems, U.S.A.) at 37 °C for 2 h. The total volume of the reaction was 50 μL, and all samples were centrifuged (3000 rpm, 30 s) before fluorescence measurement. Fluorescent spectra was recorded by a Hitachi F-4600 fluorescence spectrophotometer (Japan) under the excitation at 561 nm. To get quick measurement, a Tecan Infinite M-200 plate reader (Switzerland) was used in quantification tests (Ex = 561 nm, Em = 627 nm, 25 reads per sample). B


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Analytical Chemistry 2.4. Gel Electrophoresis. Native-PAGE (12%) gel was used in gel electrophoresis. Samples were mixed with 5× loading buffer (containing 20% ficoll) and loaded in a volume of 30 μL and ran in 0.5× TBE buffer at 80 V for 2.5 h. After staining with 4S Red Plus nucleic acid stain (Sangon Biotech, Shanghai, China) for 1 h, gel image was photographed by 3500R Gel Image System (Tanon, Shanghai, China). 2.5. Cell Culture and Treatment. Human colon cancer DLD-1 cells obtained from the American Type Culture Collection were maintained in RPMI-1640 supplemented with 10% fetal bovine (Gibco, U.S.A.). The cells were cultivated in the incubator (Sanyo, Japan) with 5% CO2 at 37 °C. In the H2O2 and TNF-α treatment experiments, DLD-1 cells were incubated with H2O2 (Basic Bioscience Inc., Shanghai, China) or TNF-α (PeproTech, Rocky Hill, NJ) for 30 min before harvesting. The nuclear extracts were harvested by a nucleoprotein extraction kit (Sangon Biotech, Shanghai, China) following manufacturer’s instructions. Before quantification of TFs, the total protein concentration of all samples was tested and unified using a Bradford-based test kit (Beyotime, Shanghai, China).

Scheme 1. Schematic Illustration of This Analytical Method

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RESULTS AND DISCUSSION 3.1. Principle of Exo III-Assisted Amplification Detection of Transcription Factors Using AgMBs. This method is based on two major building blocks, which are Exo III and AgMBs. Exo III helps to translate the protein binding into ssDNA releasing as well as amplify the detection signal, while AgMBs produces changeable fluorescent signal for quantification. Exo III exhibits 3′ to 5′ exodeoxyribonuclease activity specific for blunt ends and 5′-overhang ends dsDNA.48 Besides, the binding of TF to its binding site can prevent the dsDNA from Exo III digestion because the digestion of this enzyme cannot proceed past the TF−DNA complex.49,50 AgMBs has a nucleation sequence at 5′ ends to form DNAAgNCs and a G-rich enhancer sequence (GRS) at the 3′ ends. When AgMBs are in stem-loop structure, the proximity of DNA-AgNCs and GRS causes fluorescence enhancement, whereas the hybridization of AgMBs and complementary DNA causes a fluorescence decrease because the opening of the stem-loop structure leads to the GRS moving away from DNA-AgNCs.42,46 As exemplified in Scheme 1, our detection strategy can be divided into two parts, including signal translation (light blue backgrounded) and signal detection (pale orange backgrounded). Generally speaking, the binding of TFs is translated into ssDNA releasing, and then the released ssDNA is detected. The ssDNA is a bridge connecting the two parts, so it is referred to as the reporter. The dsTFs probe (1) contains a TFs binding area (red region) and a sequence partially complementary to AgMBs (blue region). This blue colored sequence is set at the 3′-overhang ends side to antagonize Exo III digestion. In the presence of target TFs, the TFs specifically bind to the binding area to form a TFs-DNA complex and protect the dsDNA from digestion. So, no reporter can be released and high fluorescence can be detected. However, in the absence of TFs, Exo III degrades dsTFs probe and releases the reporter. Then the reporter hybridizes with AgMBs to form a partial double-stranded intermediate (2), which causes the opening of the stem-loop structure and a fluorescence decrease. Meanwhile, Exo III digestion consumes the GRS (green region) from the blunt 3′ ends of the intermediate, causing the release of low-fluorescent DNA-AgNCs (3) and intact reporter (4). Then

the reporter functions as a template to hybridize with the remaining AgMBs for the next cycle to trigger an Exo III recycling amplification reaction. This recycling reaction pumps the transformation of AgMBs from a hairpin-shaped highly fluorescent configuration to a GRS-removed weakly fluorescent form, resulting in a significant fluorescence decrease. In this research we mainly used NF-κB p50 as a model TF, so the dsTFs probe was named as dsNF-κB p50 probe in the following sections. 3.2. Optimization and Verification of Detection Strategies. AgMBs were synthesized according to previous reports.46 Upon being excited at 571 nm, it revealed fluorescence at 627 nm, which is in agreement with former literature.40,46 As indicated in Figure 1A, the addition of a complementary reporter DNA caused the fluorescent decrease of AgMBs. Wang’s group has utilized this phenomena to detect virus genes with a limit of detection (LOD) of 8.5 nM, which proved the sensitivity and selectivity of this reaction.46 To further polish up the sensitivity of this method, we introduced the Exo III recycling amplification into this reaction. As shown in Figure 1B, an identical concentration of reporter DNA generated a greater signal decrease than untreated ones. Furthermore, no great change can be observed in the black solid curves in Figure 1A,B, which means that Exo III and the reaction buffer had negligible influence on the stability of AgMBs. Thus, the utilization of Exo III can significantly increase the sensitivity for sensing reporter. To avoid the waste of TFs (which is quite expensive), we started with optimizing the dosage of Exo III, incubation time and the sequence design of reporters. As shown in Figure S1A, high dose of Exo III can not only cause DNA-AgMBs respond stronger to the reporter but also quench the fluorescence of DNA-AgMBs slightly. When taking the fluorescence change ratio (I/I0) into consideration, 20 U of Exo III was adopted for further investigation. Afterward, incubation time was optimized C


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Figure 1. (A) Fluorescent spectra of AgMBs after addition of different concentrations of reporter DNA. In total, 10 μL of 1 μM AgMBs was mixed with 1 μL of different concentrations of reporter and 9 μL of DEPC water, then incubated at 37 °C for 1 h. (B) Fluorescent spectra of AgMBs after addition of Exo III and reporter DNA. Experimental conditions of this test were the same as (A), except for DEPC water was replaced by 10 U of Exo III and Exo III buffer.

(Figure S1B). In consideration of the fluorescence change ratio (I/I0), 120 min was chosen as the optimized incubation time. As for the design of reporter, we synthesized several variants with 27 to 35 bases complementary to the AgMBs (sequences listed in Table S1). Due to the difference in binding energy, the longer the complementary reporters are, the stronger the signal response can be reached (see Figure S2). However, longer reporters lead to more incubation time and higher cost. According to the amplification ratio (Ra), reporter-6 was chosen as the optimized sequence (Figure S1C). Then, an sreporter-6 was synthesized and hybridized with reporter-6 to form dsNF-κB p50 probe. To verify the feasibility of our detection strategy, we used native-PAGE to monitor the analytical process. As a label-free method, the effect of DNA-AgNCs to the templet DNA should be a great concern because the nucleation may cause the change of templet DNA’s secondary structure. A previous report has demonstrated that the DNA-AgNCs can little affect the conformation of templet DNA, but the stem-loop structure remains unchanged.42 Our research showed that the sample of AgMBs and templet DNA had stripes with similar migration speed (Figure S3A), which supports the previous report.42 Furthermore, the stripe of Lane 2 in Figure S3A was cut and soaked in buffer to confirm its composition, and the eluate exhibited similar fluorescent features with AgMBs (Figure S3B). Afterward, several committed steps are recorded and shown in Figure 2. Lane 1 (s-reporter-6), Lane 2 (reporter-6), and Lane 5 (AgMBs) were used as markers. The hybridization of s-reporter-6 and reporter-6 led to a bright dsNF-κB p50 probe stripe (Lane 3). After mixing the dsNF-κB probe with Exo III and incubating for 30 min, a single stripe with the same migration speed as reporter-6 can be achieved (Lane 4). The single stripe of Lane 4 indicated the Exo III digestion was fully proceeded. The mixture of AgMBs and reporter-6 produced a new dsDNA stripe in Lane 6, which was the partial doublestranded intermediate ((2) in Scheme 1). Lanes 7−9 were the confirmation of the signal amplification reaction. The dsNF-κB probe was first digested by Exo III for 30 min, then mixed with AgMBs for 30 min (Lane 7), 60 min (Lane 8), and 90 min (Lane 9). It can be observed that the stripes of AgMBs became weaker, and they were digested into short DNA fragments with

Figure 2. Verification of the analytical process by gel electrophoresis. Lanes 1, 2, and 5 were 1 μM of pure s-reporter-6, reporter-6, and AgMBs, respectively. Lane 3: 1 μM of dsNF-κB p50 probe. Lane 4: 1 μM of dsNF-κB p50 probe incubated with 20 U of Exo III for 30 min. Lane 6: 1 μM of AgMBs mixed with 0.2 μM of reporter-6. Lanes 7−9: 0.1 μM of dsNF-κB p50 probe incubated with 20 U of Exo III for 30 min, then 1 μM of AgMBs were added and reacted for 20 min (Lane 7), 40 min (Lane 8), and 60 min (Lane 9), respectively. Lane 10: 1 μM of dsNF-κB p50 probe incubated with 1.5 μM NF-κB p50 for 30 min, then 20 U of Exo III were added to digest for 30 min, followed by 60 min of reaction with 1 μM of AgMBs. Total volume of each sample were 20 μL, and all incubations were performed at 37 °C.

the extension of reaction time. It is noteworthy that only little dsNF-κB p50 probe were added to the samples of 7−9, so no distinct stripes of reporter-6 and partial double-stranded intermediate can be observed due to the limited resolution of gel stain. In Lane 10, only a band of AgMBs and a slowmigrated stripe can be recorded. According to previous reports, the slow-migrated stripe was the complex of NF-κB p50 the dsNF-κB probe,27,51 which could be cut and eluted for further TFs-related research.11 Importantly, it is clear that no reporter6 can be released and AgMBs remained unchanged in the sample of Lane 10, which indicated that the digestion of dsNFκB probe was blocked in the presence of TFs. Notably, sample addition in this test (detailed in legends of Figure 2) did not follow the detection protocol in the Experimental Section to get good visibility, while this test can still visually confirm the feasibility of our method. A group of competitor dsDNA were designed to further confirm the veracity and specificity of out detection assay. D


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comparative study between these competitor groups, it can be confirmed that our detection is specific to initial dsNF-κB p50 probe, which reinforces the veracity of this assay. 3.3. Detection of Pure NF-κB p50. To demonstrate the performance of our method, various concentration of NF-κB p50 were added to the reaction system. The fluorescent reads against the concentration of target are shown in Figure 4A. It can be observed that the enhancement of fluorescence was directly proportional to the concentration of NF-κB p50 within the linear range (30 pM−1.5 nM). The linear curve of our detection method was consistent with the nature of Exo IIIbased linear amplification strategies. An LOD of 10 pM can be achieved (S/N = 3, detailed in Supporting Information). The sensitivity has improved by 4 orders of magnitude compared with FRET-based method1 and by 3 orders of magnitude compared with ruthenium complex-based method.30 The high sensitivity is attributed to the signal amplification as well as the high signal/noise features of AgMBs.40 It should be noticed that the linear range and LOD can be adjusted by changing the amount of dsNF-κB p50 probe, and the above-mentioned analytical performance was the result of balancing sensitivity and our needs for detecting cell samples. We chose bovine serum albumin (BSA, widely used in molecular biology research) and four human proteins including human serum albumin (HSA), insulin, and NF-κB p65 as model interferent to test the selectivity of our method. As provided in Figure 4B, when 0.5 nM of all these proteins were added to the reaction, only NF-κB p50 caused strong signal response. NF-κB p65 is a TFs in the NF-κB family with different binding site is different from NF-κB p50. Because of the sequence-specific binding nature of TFs, only NF-κB p50 could efficiently bind to the dsNF-κB p50 probe and prevent the probe from Exo III digestion. This results revealed that our method has a good selectivity toward NF-κB p50. 3.4. Detection of Endogenous TFs. The ability of our method to detect endogenous TFs was investigated using DLD-1 cells as a testbed. We began with investigating a recovery test. We substituted the fluorescent reads of unspiked samples into the linear equation and assumed that the corresponding concentration data (99.12 pM) was the concentration of this sample. Then, the samples were spiked with different amount of pure NF-κB p50 and quantified by our method. As shown in Table S2, the recovery was basically distributed around 100% when spiked with NF-κB p50 in the linear range. We organized these data in Figure 5A. It can be observed that all the recovery were distributed in the acceptable range for bioanalysis (80−120%), and a wide range of this data were fallen in the ideal range for recovery (90−110%) in an analytical procedure. Therefore, the above-obtained linear equation can be adopted in real sample analysis with reasonable errors. Then, we investigated the practical application of our method by comparing the concentration of endogenous NF-κB p50 in untreated DLD-1 cells with treated ones. Two groups of treated DLD-1 cells were set in this investigation, and they were incubated with H2O2 and TNF-α respectively. H2O2 and TNFα are stimulators to increase the NF-κB p50 level in cell nucleus. Besides, a deactivated group was set by deactivating nucleoprotein of DLD-1 cells at 75 °C. The results of this test were shown in Figure 5B. In the deactivated group, the cause of low fluorescent signal was that deactivated proteins was unable to prevent the release of reporter-6. Conversely, the other three groups all showed distinct fluorescent signal due to the binding

Competing probe (cP) is a short dsDNA with NF-κB p50 binding site but contains no efficient sequence to hybridize with AgMBs. In contrast, random probe can complement to AgMBs, but a random sequence is substituted for the NF-κB p50 binding sequence. S1−S4 are designed as four analogues of dsNF-κB p50 probe with one to four nucleotides replaced in NF-κB p50 binding site. Figure 3 shows the sequence design

Figure 3. Verification of the specificity of this method. The red letters denote the binding site of NF-κB p50, while the gray letters denote the substituted nucleotide in the binding site. The peach lines in competing probe (cP) represent that the remaining sequence is different from others (who are colored with black) and they cannot complement with AgMBs (sequence detailed in Table S1). Error bars indicate the standard deviation of six replicate determinations. Experimental details: 100 nM of these probes were mixed with 1.5 nM NF-κB p50 and analyzed following the protocol described in the Experimental Section.

and test results (Table S1 can be consulted for whole sequences). The addition of NF-κB p50 caused significant fluorescence enhancement in the dsNF-κB p50 probe group because the binding between NF-κB p50 and dsNF-κB p50 probe efficiently blocked the release of reporter-6. In contrast, the samples with and without NF-κB p50 in the cP group both exhibited high fluorescence. Although ssDNA can be released from the sample of cP group without NF-κB p50, but the released ssDNA do not have the sequence to hybridize with AgMBs. In the case of S1−S4, on the one hand, the absence of NF-κB p50 caused low fluorescence as speculated, on the other hand, the fluorescence enhancement caused by NF-κB p50 reduced with more nucleotides in the NF-κB p50 binding site are substituted. Besides, rP can release reporter-6 both with and without the existence of NF-κB p50, so weak and similar fluorescent intensity was recorded in this group. By making a E


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Figure 4. (A) Relationship between the fluorescent intensity and the concentration of NF-κB p50. (B) Fluorescent intensity of the reaction when 0.5 nM of different proteins were added to the initial system. Data were collected following the protocol described in the Experimental Section, and error bars indicate the standard deviation of six replicate determinations.

Figure 5. (A) Recovery of NF-κB p50 in DLD-1 nucleoprotein extracts. (B) Fluorescent intensity of the reaction system when the DLD-1 cells were treated differently. Experimental details: (A) 5 μL of nucleoprotein extracts were mixed with various concentration of pure NF-κB p50 and analyzed according to the protocol; (B) 10 μL of differently treated nucleoprotein samples were analyzed. Error bars indicate the standard deviation of six replicate determinations.

proteome scale like mass spectrometry-based methods,53 its ability to monitor certain critical TFs with simple pretreatment and low cost still makes it a promising tool for practical application. It should be noticed that p53, AP1, CREB, and TBP were analyzed following the protocol of NF-κB p50. To get more accurate results, optimization for each targets should be done according to the steps discussed above. 3.5. Inhibition Assay. NF-κB plays important roles in a wide range of diseases, and the constitutively active of NF-κB has been found in many cancers.24 Thus, developing strategies for blocking NF-κB activation has become a major target in drug development. NF-κB activation can be suppressed by blocking various steps in its signaling pathway, but the most direct strategy is to block the binding of NF-κB to DNA.55 In principle, an efficient analytical method for measuring TFs can be turned into a platform for screening potential drugs. Therefore, we investigated the capability of our method to screen inhibitory compound by fixing the concentration of NFκB p50 at 1.5 nM. We chose (−)-DHMEQ as a model inhibitor which inhibits the binding of NF-κB p50 to DNA by covalently

between active NF-κB p50 and dsNF-κB probe. When comparing the H2O2 treated and TNF-α treated groups with the untreated group, a 1.68-fold and a 2.41-fold of signal increase can be observed, respectively. This increase was in accordance with previous knowledge,11,21,52 which indicated the ability of our method to detect NF-κB p50 in real samples. Furthermore, we implement this assay to measure other TFs to ascertain whether our detection system have generality in TFs detection. DLD-1 cells were treated with TNF-α for 15, 30, and 60 min. Then, by replacing the TFs binding site in dsTFs probe, we quantified several representative endogenous TFs. Figure 6 depicts the time-dependent level change of NFκB p50, p53, AP1, CREB, and TBP. The regulation profile provided by our method is basically consistent with former literatures,11,53,54 which indicates that our method can be used as a versatile platform to detect different TFs. Besides, by analyzing samples in 96-well plate and organizing data into a heat map (as shown in Figure 6), our method can quickly conduct TFs profiling analysis toward multiple TFs. Though it is difficult for our method to provide a global TF alteration at F



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CONCLUSION The changeable fluorescence of AgMBs are modulated by a digestible DNA sequence, which made it suitable for involving in an enzyme-assisted amplification reaction. On this basis, we integrated AgMBs with Exo III to develop a homogeneous method for fluorimetric detection of TFs. The present method could quantify NF-κB p50 with a 10 pM detection limit and a wide linear range over 3 orders of magnitude under isothermal conditions, and only two steps of reagent addition were needed. Then, the detection ability of our method was verified by detecting multiple endogenous TFs in DLD-1 cells. Moreover, by mixing fixed concentration of NF-κB p50 with various concentration of (−)-DHMEQ, we testified the drug-screening aptitude and IC50 calculation capability of our method. Taken together, this study not only enlarged the application range of DNA-AgNCs materials by first using AgMBs in TFs detection, but also provided a highly promising analytical technique for TFs-related biomedical researches.

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Figure 6. Detection of multiple TFs in DLD-1 cells. Cells are treated with TNF-α for 15 min (red columns), 30 min (blue columns), and 60 min (orange columns). The heat map shows the relative amount of TFs compared with 0 min group, and the green to red represent down-regulation and up-regulation, respectively. Data were collected by analyzing 10 μL of nucleoprotein samples following the protocol we established, and error bars indicate the standard deviation of three replicate determinations.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b00055. DNA sequences used in this work; recovery of NF-κB p50 in DLD-1 nucleoprotein extracts; optimization of analytical conditions and reporter; gel image of AgMBs and its DNA template; calculation of LOD (PDF).

binding to Cys residue of Rel family proteins.56 The analytical process was conducted after NF-κB p50 (1.5 nM, 5 μL) were mixed with 5 μL of different concentration of (−)-DHMEQ. The fluorescent signal against the concentration of (−)-DHMEQ are shown in Figure 7, and it can be found

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 25 86868476. Fax: +86 25 86868476. E-mail: [email protected]. ORCID

Xuemin Zhou: 0000-0001-7717-0436 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 81572081, 81273480, 21175070, and 81202853) and Natural Science Foundation of Jiangsu Province (BK2012444).

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REFERENCES

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Figure 7. Inhibition effect of (−)-DHMEQ measured by our method. The concentration of NF-κB p50 was fixed at 1.5 nM and then incubated with different concentration of (−)-DHMEQ. Error bars indicate the standard deviation of six replicate determinations.

that the fluorescence decreased along with the addition of (−)-DHMEQ. Further, the half-maximal inhibition concentration (IC50) of (−)-DHMEQ was calculated as 12.24 nM by fitting the raw data. As a homogeneous assay, our method has the aptitude for high-throughput screening inhibitors of target TFs in the compound library at the molecular level and calculating the IC50 of corresponding candidates. G


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